9°
BIOCHIMICA ET BIOPHYSICA ACTA
ON T H E S T R U C T U R E
VOL. 8 (1952)
OF THE SPINACH CHLOROPLAST
by
J. B. THOMAS*
Biophysical Research Group Uteeeht-Del/t, under the Direction of A. J. KLIdYVER, Del#,
and o] J. M. W. MILATZ, Utrecht (Netheylands)
and
M. BUSTRAAN AND C. H. PARIS
Physical Institute, University, Utrecht (Netherlands)
INTRODUCTION
Many investigations on the structure of chloroplasts have already been carried out.
F o r reviews we refer to WEIER 1, FREY-WYsSLING 2, 3, RABINOWITCH4, AND GRANICK5.
Results obtained with the light microscope as well as studies on birefringence have
provided valuable information. These data and deductions have been partly checked
b y experiments with the electron microscope which has made it possible to observe and
to photograph submicroscopic- and even some amicroscopic structures.
Summarizing the main results emerging from a study of the literature one can state
that the chloroplast:
1. is surrounded by a membrane (KNuDSON 6, ALGERA e~ al. 7, GRANICK AND PORTERs,
FREY-WYsSLING AND MUHLETHALER°).
2. consists of a colourless stroma in which green grana are embedded. This holds
at least for various species (MEYER 1°, SCHIMPER11, DOUTRELIGNE12, HEITZ 13, GEITLER 14,
ROBERTSIS). The occurrence of grana has often been doubted. This was due firstly to
the fact that ill the light microscope chloroplasts of some species show a granular
appearance only after swelling, e.g., in distilled water. Secondly, in other species grana
are never observed. However, due to the high resolving power of the electron microscope
this question has been settled. Whereas the visibility of the grana under the light
microscope is restricted to those with a diameter surpassing 0.3 t~, their occurrence has
now been proved in all cases investigated.
3- m a y contain pyrenoids or starch grains (el. MENKE TM, RHOADES AND CARVALHO17).
So far, however, the picture of chloroplasts of even one species is only incomplete.
In the study presented here some more data are given.
MATERIAL AND METHODS
Washed leaves of Spinacia oleracea in o.i 5 M phosphate buffer--pH 6.o--were grinded in a
"Waxing blendor" during 5 minutes. The suspension was filtered through cotton wool and centrifuged
This investigation has been made possible by a grant from the Netherlands Organisation tor
Pure Research (Z.W.O.).
Re]erences p. IOO.
VOL. 8 (1952)
STRUCTURE OF THE SPINACH CHLOROPLAST
9I
at an acceleration of a b o u t 1. 3 • lOs cm/sec 2 during 5 minutes. The green u p p e r layers of the s e d i m e n t
were carefully r e m o v e d and transferred into a IO~o saccharose solution. The suspension was washed
b y centrifuging a n d r e s u s p e n d i n g 2 - 3 times. Our t h a n k s are due to Miss L. J. BARTELS for preparing
several batches of chloroplasts in this way.
The p r e p a r a t i o n s were m o u n t e d in the usual manner. The 3, were studied in a Philips electron
microscope. S h a d o w casting was performed with a gold-manganin m i x t u r e (i : I).
Extraction o/ the lipoids by means o/ benzene was carried out by m o u n t i n g the chloroplasts on
the p r e p a r a t i o n slide and b y adding a drop of benzene, after drying the m o u n t , renewing the drop
if necessary. The d u r a l i o n of the extraction varied from 3-2o minutes.
Removal o/the lipoids by means o~ lipase was effected in different ways. The enzyme p r e p a r a t i o n
- - c o n s i s t i n g of a concentrated p a n c r e a t i n s o l u t i o n - - w a s acidified by means of diluted HC1 to a PH
of a b o u t 6.0. I n this m a n n e r the activity of t r y p s i n was inhibited, whilst t h a t of lipase was only
p a r t l y reduced. I n some cases the chloroplasts were fixed with 2 or IO% osmic acid solution.
The reagents were applied in different succession as follows:
I. Mounting of the chloroplasts, followed by enzyme addition; 2. fixation with osmic acid,
mounting, e n z y m e addition; 3. mounting, fixation, enzyme addition; 4- enzyme addition, m o u n t i n g ;
5- e n z y m e addition and fixation, mounting.
If digestion had proceeded to a large extent, fixation of the material with osmic acid proved
to be desirable. However, the succession of the addition of the reagents t u r n e d out to be of m i n o r
importance.
The enzymic t r e a t m e n t took place at 37°C during 15-4o minutes. If the chloroplasts were
m o u n t e d before digestion the droplet of the enzyme solution had to be renewed several times.
Removal o/ the p~oteins was effected by using a highly purified pepsin p r e p a r a t i o n of PH 4 .o.
F o r particulars we refer to the above mentioned description of the lipase procedure. We wish to
express our gratitude : o w a r d s Dr J. C. M. MIGHORST, who kindly placed the enzyme p r e p a r a t i o n s
at our disposal.
Ultrasonic treatment was performed in a magnetostriction oscillator. The energy delivered by
the magnetostrictive t u b e was a b o u t 3o0 watts, at a frequency of 700o Herz. A p p r o x i m e t e l y iO~o
of the energy was effective. The chloroplasts -*'ere t r e a t e d for I, 4, and 6 minutes. I n the latter case
the oscillator cup was filled with the chloroplast suspension, whilst in the experiments of shorter
d u r a t i o n a test-tube cc.ntaining the chloroplasts was a t t a c h e d to the vibrating cup. :By the courtesy
of Dr J. A. NIEMEYER these e x p e r i m e n t s could be carried out with the oscillator of the L a b o r a t o r y
for Physiological Chemistry at Utrecht.
RESULTS AND DISCUSSION
On the constitution o[ the chloroplast membrane
Fig. I represenr.s a chloroplast after lipoid extraction with benzene. Since a folded
bladder can be clearly seen, it must be concluded that proteins are an important constituent of the membrane. If, however, the proteins are removed by pepsin digestion
(Fig. 2) only remnants of the membrane, composed of lipoids, are to be seen. These
remnants seem to b,~ very fragile, since they are always torn to pieces. It is tempting
to draw a parallel with the erythrocyte membrane, which--according to WINKLER AND
BUNGENBERG DE JONGls, and to JUNG 1 9 - is composed of a lipoid outer l a y e r - - e p i l e m m a
- - a n d a protein inner layer. The proteinaceous layer is supposed to function as a bearer
for the delicate lipoid film.
On the constitution o/the stroma
Figs 3 and 4 show a part of a chloroplast after lipoid extraction with benzene. The
proteinaceous framework can be seen between the seemingly intact grana. This is in
agreement with the results of GRANICK AND PORTER. If, however, the proteins are
digested b y pepsin, the remaining structure is quite different (Figs 5 and 6). The sharp
edges of the grana have disappeared, as demonstrated by the remnants of the grana
Re/erences p. zoo.
92
J. 13. THOMAS, M. BUSTRAAN C. H. PARIS
VOL. 8 (I952)
Fig, I. Proteinaceous c o m p o n e n t of the chloroplast m e m b r a n e . Benzene extraction. Shadowed. ~oo kV
Fig. 2. Lipoid c o m p o n e n t of the chloroplast m e m b r a n e . Pepsin digestion. Shadowed. ioo kV
VOL. 8
(I952)
STRUCTURE OF THE SPINACH CHLOROPLAST
Fig. 3- Proteinaceous c o m p o n e n t of the s t r o m a and grana. Benzene extraction, too kV
Fig. 4. See Fig. 3. Shadowed. 8o kV
Fig. 5. Lipoid c o m p o n e n t ot the s t r o m a and grana. Pepsin digestion. 8o kV
Fig. 6. See Fig. 5- Shadowed. Ioo kV
93
94
j . B . THOMAS, M. BUSTRAAN, C. H. PARIS
VOL. 8 (I952)
I
Fig. 7. Membranes with macromolecular structure in the stroma. Lipase digestion. Shadowed. ~oo kV
m a r k e d X. I n s t e a d of a f r a m e w o r k an a m o r p h o u s m a s s of lipoids r e m a i n s . I n this m a s s
o p e n s p o t s are a l w a y s found. P o s s i b l y t h e y r e p r e s e n t holes r e m a i n i n g a f t e r t h e r e m o v a l
of t h e p r o t e i n a c e o u s fibrils.
W h e n the lipoids are d i g e s t e d b y lipase a n d fixed w i t h osmic acid, m e m b r a n e s of
a b o u t IOO A t h i c k n e s s are o b s e r v e d . T h e s e m e m b r a n e s s h o w a m a c r o m o l e c u l a r design
(Fig. 7). T h e r e is a s t r i k i n g r e s e m b l a n c e to p i c t u r e s p u b l i s h e d b y FERNANDEz-MoRAN 20
a n d r e p r e s e n t i n g t r a n s v e r s e sections of t h e m y e l i n s h e a t h of t h e n e r v u s i s c h i a d i c u s of
t h e a l b i n o rat. I n our case, h o w e v e r , we are d e a l i n g w i t h s u r f a c e s t r u c t u r e s . T h e t h i c k ness of t h e fibrillae a m o u n t s to IOO 15o A, whilst for t h e i n t e r f i b r i l l a r s p a c e a b o u t 5 ° A
was m e a s u r e d .
After prolonged
4 ° minutes
lipase d i g e s t i o n t h e s e m e m b r a n e s d i s a p p e a r e d .
P r o b a b l y t h e y b e c a m e t o o d e l i c a t e to r e m a i n i n t a c t a f t e r t h e t o t a l r e m o v a l of lipoids.
A p i c t u r e as r e p r e s e n t e d b y F i g s S a n d 9 is o b t a i n e d . I n t h e s e e x p e r i m e n t s no osmic
a c i d f i x a t i o n was p e r f o r m e d in o r d e r to e n s u r e m a x i m a l lipase a c t i v i t y , w h i l s t f i x a t i o n
of a l r e a d y m o u n t e d m a t e r i a l s e e m e d superfluous, if n o t h a r m f u l , for t h e fragile s t r u c tures. H o w e v e r , t h e p o s s i b i l i t y of o b t a i n i n g a r t e f a c t s r e m a i n s . B u t a c o m p a r i s o n w i t h
e l e c t r o n - m i c r o g r a p h s p u b l i s h e d b y o t h e r a u t h o r s (e.g. LEHMANN AND B l s s 21) m a k e s us
b e l i e v e t h a t h e r e too we are d e a l i n g w i t h n a t u r a l l y o c c u r r i n g p r o t e i n s t r u c t u r e s . F o r
r e v i e w s on this s u b j e c t we refer to MONNg: ~2 a n d RONDONI 2a.
Cytoplasmic fibrils are described as being composed of tiny globules "Chromidia" or "Bios o m e n " - - w i t h a diameter of o. ~ 0.2 #, and linking t h r e a d s - - " I n t e r c h r o m i d i a " --, just like the genes
in a chromosome. The globules contain phospholipids, ribonueleic acid and a large amount of calcium.
They are said to be autoreproductive and to represent "centres" for respiration and growth. In our
objects we tried to show a diiterence between the composition of the "globules" and the "thread"
as follows. If it were trne that here too the "globules" contain metals, e.g., calcium, we may expect
that, per unit of thickness, they cause a stronger electron scattering in the microscope than the
"thread" does. In a shadow-casted preparation we determined the ratio (a) length of "globule"
shadow/length "thread" shadow. [n a preparation without shadowing we measured the light transmission of "globules" and "threads" in a photometer. From the records the ratio (b) absorption by
Re/erences p. loo.
VOL. 8 (1952)
d
STRUCTURE OF THE SPIN~\CH CHLOROPLAST
!
,
95
I
Fig. 8. Proteinaceous framework of the stroma. Lipase digestion. 8o kV
Fig. 9. See Fig. 8. Shadowed. ioo kV
o
9 {)
J.B. THOMAS, M. BUSTRAAN, C. H. PARIS
VOL. 8 (I952)
"globules"/absorption by "thread" was determined. Among the values for the ratio (b)/(a) we
obtained figures up to 5.9. This offers a strong indication that the "globules" contain a substance
which causes a larger electron scattering than the "thread" material does, and which for instance may be metals.
This is, of course, only a preliminary experiment. To obtain more accurate data it is necessary
to study the same preparation before and after shadow-casting and to appl~c corrections with regard
to the blackening curves of the photographic material.
Spinach chloroplasts have been c h e m i c a l l y a n a l y z e d b y several a u t h o r s (c/. RABINOWlTCH~). The percentages of proteins v a r y from 39.6 to 58, those of lipoids from 25.I
to 34, a n d those of t h e rest from IO to 35.3. The l a t t e r values include c a r b o h y d r a t e s .
I t should be m e n t i o n e d here t h a t we e m p l o y e d starch-free chloroplasts. We (lid not
notice " v a c u o l e s " in which the starch could be deposited. However, it might be possible
t h a t the pieces of m e m b r a n e shown in Fig. 7 belong to such a vacuole wall. \Ve are not
y e t certain on this point.
S u m m a r i z i n g we o b s e r v e d the following d a t a concerning the s n o m a :
I. occurrence of a p r o t e i n a c e o u s frame in which the g r a n a are einbedded.
2. this frame consists of " g l o b u l e s " linked t o g e t h e r b y a "thr(~ad"
Chromidia a n d
I n t e r c h r o m i d i a - - . The "globules" c o n t a i n substances with a larger electron s c a t t e r i n g
power t h a n those of the " t h r e a d " .
3. occurrence of i n t r a c h l o r o p l a s t i c m e m b r a n e s with m a c r o m o l e c u l a r surface structure.
4. after r e m o v a l of t h e p r o t e i n s a " s p o n g y " lipoid mass remains. The holes m a y
represent channels originally filled b y t h e p r o t e i n a c e o u s fibrils.
On the constitution o/tl~e grana
I n a g r e e m e n t with d a t a m e n t i o n e d in l i t e r a t u r e (cf. GRANICK AND PORTER s) the
spinach g r a n a are w a f e r - s h a p e d discs with a d i a m e t e r of a b o u t 0. 5/x a n d a b o u t 0.07/~
thick. Often the parallel surfaces show a central swelling. After t r e a t m e n t with benzene
a n d lipase the edges of the g r a n a are just as d i s t i n c t l y visible as t h e y are in u n t r e a t e d
condition, c/. Figs 3 a n d 4- If, however, the p r o t e i n s are removed, a quite different
p i c t u r e is obtained. This is d e m o n s t r a t e d in Fig. 5 and Fig. IO. In the l a t t e r figure
lipoid films which show the a p p r o x i m a t e form of the g r a n a can be seen. The delicateness
of these films is i n d i c a t e d b y the fact t h a t t h e y are not visible in u n s h a d o w e d p r e p a r a t i o n s (Fig. 5).
F r o m these d a t a we conclude t h a t the m e m b r a n e s of the g r a n a are also composed
of at least two c o m p o n e n t s : p r o t e i n s a n d lipoids. F u r t h e r m o r e we o b t a i n e d the impressior) t h a t these substances are a r r a n g e d in two layers, the lipoids forming the outer
layer. This is d e d u c e d from Fig. I I , which gives a result which is often found after lipoid
e x t r a c t i o n , in this case with benzene. Many g r a n a are d e t a c h e d from the stroma, leaving
r o u n d holes behind. These holes have a l r e a d y been described b y W I E L E R 2"1, who obserw~d
t h e m in chloroplasts e x t r a c t e d with alcohol a n d concluded t h a t the g r a n a are c o m p o s e d
of f a t t y substances. A p p a r e n t l y the d e t a c h e d g r a n a could not be seen u n d e r the light
microscope. Of course, it does not follow from Fig. i i t h a t lipoids c o n s t i t u t e the outer
layer of the grana. I t m a y be possible t h a t the g r a n a are e m b e d d e d in lipoids belonging
to the stroma. However, as m e n t i o n e d above, pictures as those represented in Fig. I o
are in favour of the view t h a t lipoids compose the outer layer of the g r a n a r m e m b r a n e .
According to FREY-\VYSSLING AND MI)HLETHALER ~ the g r a n a are c o n s t t u c t e d of
a l t e r n a t i n g protein- and lipoid lamellae. In one of their pictures the' protein lamellae are
Re/erences p. [oo.
VOL. 8
( z9 5 2 )
STRUCTURE OF THE SPINACH CHLOROPLAST
97
Fig. io. Lipoid c o m p o n e n t of tbe g r a n a r membrane. Pepsin digestion. Shadowed. ioo kV
Fig. i i . Evidence of a lipoid outer layer of the granar membrane. Benzene extraction. Shadowed.
ioo kV
Fig. 12. G r a n a r lamellae. 4 m i n u t e s ' supersonic t r e a t m e n t . Shadowed. 8o kV
Fig. 13. G r a n a r lamellae and myelin formation. 4 m i n u t e s ' supersonic t r e a t m e n t . Shadowed. 8o kV
Fig. i t. Small g r a n a r lamellae. 6 m i n u t e s ' supersonic t r e a t m e n t . Shadowed. 8o kV
9g
j.B. THOMAS, M. BUSTRAAN, C. H. PARIS
VOL. 8 (1952)
clearly shown. It was deduced from their intact form that they must be protein layers
since lipoid layers should have combined to form myelin figures. The protein lamellae
are shown also in the paper of GRANICK AND PORTERs (their Fig. 5) although they are
not recognized as such.
We succeeded in dislodging these lamellae by brief exposures I to 4 m i n u t e s - to ultrasonic disintegration of the grana (Fig. I2). In some cases, as is shown for instance
in Fig. I3, the lamellae are found in the presence of myelin figures which m a y be due
to fusing of lipoids in the grana. Sometimes one or two lamellae are much smaller
- - d o w n to about ~ s i z e - - t h a n the others of the same granum. It m a y be possible that
these lamellae are responsible for the previously mentioned swelling on the granar surface.
After six minutes' exposure to ultrasonic vibrations the grana seem to be thouroughly
destroyed except for these small lamellae (c/. Fig. 14). It is difficult to state this with
certainty; they m a y be fragments of larger ones, However, their circular shape makes
us believe that we are dealing here with intact units. Maybe these minute lamellae are
meant b y Miss ROBERTS25, when describing " p r i m a r y granules" which would be composed of successively smaller units down to "senary granules". In our opinion, however,
granar lamellae are not composed of sub-units, but a few seem to be smaller than the
majority.
Cylindrical organs which are built up of transverse lamellae and surrounded by
a membrane are also found in zoological material. We refer to the outer segments of the
retinal rod of the guinea pig eye as described by SJOSTRAND26. This author demonstrated
that lipoid "knobs" are situated between pairs of discs. The d i a m e t e r - - 2 . 2 / z - - o f the
discs is much larger than that of the granar lamellae. The diameter and the height of
the "knobs" are 5 ° 250 A and 7 ° A respectively. We did not succeed in demonstrating
the presence of such "knobs" in the grana beyond possibility of doubt. If they do indeed
exist, they probably are much smaller than the retinal "knobs".
Sometimes we noticed typical artefacts in preparations treated with pepsin. This
is shown in Fig. 15. In two remnants of grana fan-wise oriented lipoid rods are to be
Fig. 15. Granar lipoid artefacts. Pepsin digestion. Shadowed. ioo kV
Re/erences p. zoo.
VOL. 8
(1952)
STRUCTURE OF THE SPINACH CHLOROPLAST
99
seen. They look like regular structures, but a paper of FERNANDEZ-MoRAN on structures
in vertebrate nerves convinced us that they are artefacts. This author could state that
after trypsin digestion a structure appears which " . . . . is due to disintegration of the
ordinarily compact lamellae into composite granules consisting of 5o-Ioo A high, rod
shaped single elements, which stain intensely with osmic acid. Apparently the trypsin
digestion has divested the lamellae of certain (protein) components which bind the
lipoid particles together".
Finally we recorded electron diffraction diagrams of a chloroplast, of a piece of
isolated stroma, and of a single granum. In the stroma record no diffraction rings
occurred. The chloroplast diagram showed a ring corresponding to a distance of 1.77 A.
One ring was also visible in the granum diagrams. The corresponding distances are 2.Ol
and 2.o6 A. No rings are found in diagrams of purified chlorophyll a and chlorophyll b.
It should be remarked that the circumstances under which the preparations are made,
may influence the result. In the first place we mention the velocity of drying. So it is
difficult to interpret these data. We only can state the occurrence of distances in the
order of a few A in the grana and their absence in the stroma. They might be due to
amino acids.
Our thanks are due to Dr J. C. SCHOONEN for his kind help in evaluating the
diagrams.
Summarizing, the conclusion can be drawn that the grana are built up of:
i. an outer membrane which is most probably composed of a lipoid outer layer and
a proteinaceous inner layer.
2. protein discs--confirming the data of FREY-WYssLING AND M~)HLETHALER° with lipoids arranged in between. So far the exact arrangement of the lipoids could not
be ascertained.
SUMMARY
T h e s t r u c t u r e of s p i n a c h c h l o r o p l a s t s w a s i n v e s t i g a t e d w i t h t h e aid of t h e electron microscope.
I t h a s been e s t a b l i s h e d t h a t :
i. t h e o u t e r m e m b r a n e of t h e c h l o r o p l a s t s is c o m p o s e d of b o t h p r o t e i n s a n d lipoids.
2. t h e s t r o m a is also b u i l t u p b y t h e s e c o m p o n e n t s .
3. w i t h i n t h e s t r o m a m e m b r a n e s w i t h surface d e s i g n occur.
4. a p r o t e i n a c e o u s f r a m e - - w h i c h m o s t p r o b a b l y is n o a r t e f a c t - - o c c u r s in t h e s t r o m a . T h i s
f r a m e w o r k s t r i k i n g l y r e s e m b l e s t h a t f o u n d b y LEHMANN AND BISS21 in t h e Tubi/ex egg.
5. t h e g r a n a are s u r r o u n d e d b y a m e m b r a n e c o m p o s e d of p r o t e i n s a n d lipoids. S o m e evidence
h a s b e e n o b t a i n e d t h a t t h e s e s u b s t a n c e s o c c u r as a n o u t e r lipoid l a y e r a n d a n i n n e r proteinaceous
layer.
6. in a g r e e m e n t w i t h t h e r e s u l t s of FREY-WYSSLING AND MOHLETHALER 9, t h e g r a n a are f u r t h e r m o r e c o m p o s e d of p r o t e i n discs a n d lipoids.
7. g r a n a s h o w e d one ring in electron diffraction d i a g r a m s c o r r e s p o n d i n g w i t h d i s t a n c e s of
a b o u t 2 A.
RC-SUMCL a s t r u c t u r e des chloroplastes d ' 6 p i n a r d s a 6t6 6 t u d i 6 e ~ l ' a i d e d u microscope 6lectronique.
Nous avons montr6 que
I. la m e m b r a n e du chloroplaste e s t c o n s t i t u 6 e p a r d e s p r o t 6 i n e s et des lipoides.
2. le s t r o m a est 6 g a l e m e n t c o n s t i t u 6 p a r ces d e u x s u b s t a n c e s .
3. des m e m b r a n e s , q u i p r 6 s e n t e n t u n e s t r u c t u r e superficielle, s o n t observ~es d a n s le s t r o m a .
4. u n r6seau de prot6ines, p r o b a b l e m e n t p a s u n a r t 6 f a c t , et r e s s e m b l a n t 6 t o n n a m m e n t ~ la
s t r u c t u r e t r o u v 6 e p a r LEI~MANN ET BISS I1 d a n s l'oeuf de Tubi/ex, e s t r e n c o n t r ~ darts le st~onm.
Re/er~ces p. Ioo.
8
I00
J . B . THOMAS, M. BUSTRAAN, C. H. PARIS
VOL. 8
(I952)
5. les grains s o n t e n t o u r 6 s d ' u n e m e m b r a n e constitu6e p a r des p r o t 6 i n e s et des lipoides. Prob a b l e m e n t les lipoides s o n t f o r m e n t la c o u c h e superficielle.
6. en accord a v e c les r 6 s u l t a t s de FREY-WYsSLING ET ~MOHLETHALER9, ].eS grains c o n t i e n n e n t
e n o u t r e des d i s q u e s prot6iniques et des lipoides.
7- des d i a g r a m m e s de diffraction 61ectronique des g r a i n s isol~s p r 6 s e n t e n t u n cercle c o r r e s p o n d a n t
une distance d'environ 2 A.
ZUSAMMENFASSUNG
Mit Hilfe des E l e k t r o n e n m i k r o s k o p s w u r d e die S t r u k t u r der S p i n a t c h l o r o p l a s t e n u n t e r s u c h t .
Ergebnisse:
I. Die A u s s e n m e m b r a n d e s Chloroplasts b e s t e h t a u s P r o t e i n e n u n d Lipoiden.
2. D a s S t r o m a ist ebenfalls a u s diesen beiden K o m p o n e n t e n z u s a m m e n g e s e t z t .
3. I n n e r h a l b des S t r o m a s w u r d e n M e m b r a n e n m i t O b e r f l A c h e u s t r u k t u r b e o b a c h t e t .
4. D a s S t r o m a enthiilt ein fibrillares Eiweissgewebe, d a s w a h r s c h e i n l i c h kein A r t e f a k t ist. E i n
iflmliches Gebilde w u r d e y o n LEHMANN UND BISS 21 i m Tubi#x-Ei n a c h g e w i e s e n .
5. Die G r a n e n s i n d y o n einer a u s P r o t e i n e n u n d L i p o i d e n z u s a m m e n g e s e t z t e n M e m b r a n u m geben. E s ist wahrscheinlich, d a s s die Lipoide a n der Oberflache liegen.
6. W e i t e r e n t h a l t e n die G r a n e n P r o t e i n s c h e i b e n , welche s c h o n y o n FREY-WYSSLING UND
I~0HLETHALER9 gezeigt w o r d e n sind, u n d Lipoide.
7. E l e k t r o n e n d i f f r a k t i o n s d i a g r a m m e v o n isolierten G r a n e n zeigen e i n e n Kreis, welcher a u f einen
A b s t a n d y o n ungefAhr 2 A hinweist.
REFERENCES
1 E. WEIER, Bot. Rev., 4 (I938) 497.
2 A. FREY-WYSSLING, Protoplasma, 29 (1938) 279.
3 A. FREY-WYSSLING, Submicroscopic morphology o/protoplasm and its derivatives. E l s e v i e r P u b l . Co.,
A m s t e r d a m , N e w Y o r k (1948).
4 E. I. RAEINOWlTCH, Photosynthesis and related processes, I. I n t e r s c i e n c e P u b l . Inc., N e w York,
(1945).
5 S. GRANICK, Photosynthesis in Plants. I o w a S t a t e College Press, A m e s , (1949) 113.
* L. KNODSON, Am. J. Bot., 23 (1936) 694.
7 L. ALGERA, J. J. BEIJER, W. VAN ITERSON, W. K. H. KARSTENS, AND T. H. THONG, Biochim.
Biophys. Acta, I (1947) 517 •
8 S. GRANICK AND K. R. PORTER, Am. J. Bot., 34 (1947) 545.
9 A. FREY-WYSSLING AND I'{. MOHLETHALER, Vierteljahrsschr. Natur/orsch. Ges. Z~rich, 94 (1949).
10 A. MEYER, Das Chlorophyllkorn, Leipzig (1883).
11 A. F. W. SCmMeER, Jahrb. wiss. Botan., 16 (1885) i.
19 j . DOUTRELIGNE, Proc. Koninkl. Akad. Wetenschap. Amsterdam, 38 (1935) 886.
19 E. HEITZ, Planta, 26 (1937) 134.
14 L. GEITLER, Planta, 26 (1937) 463 •
15 E. A. ROBERTS, Bull. Torrey Botan. Club, 67 (194 o) 535.
16 W. MENKE, Naturwiss., 28 (194 o) 158.
17 M. M. RHOADES AND A. CARVALH0, Bull. Torrey Botan. Club, 71 (1944) 335.
is K. C. WINKLER AND H. G. BUNGENBERG DE JONG (ref. JUNG), Arch. n~erland, physiol., 25 (194o) 431.
19 F. JUNG, Naturwiss., 37 (195 o) 254.
a0 H. FERNANDEZ-MORAN, Exp. Cell Research, I (195o) 143.
91 F. E. LEHMANN AND R. BlSS, Rev. suisse zool., 56 (1949) 264.
92 E. MONNI~, Advances in Enzymol., 8 (1948) I.
29 p. RONDONI, Ergeb. Enzym/orsch., io (1949) 65.
94 A. WIELER, Protoplasma, 26 (1936) 295.
z* E. A. ROBERTS, Am. J. Bot., 29 (1942) i6s.
25 V. S. SJ6STRAND, J. Cell. Comp. Physiol, 33 (1949) 383 .
27 H. FERN/LNDEZ-MORAN, Exp. Cell Research, I (195 o) 3o9 .
Received
March
15th, 1951